1. Technical Field
The present invention is related to an apparatus and a process for fusion of cell membranes. More particularly, the present invention is related to an improved device and a method of inducing fusion of cell membranes under the influence of an electric field.
Fusion is defined as the merger or coalescence of at least two cell membranes, spaces or cavities to form a single cellular entity. Fusogenic state is defined as an altered, storable state of the membranes induced by first exposing the membranes to a direct current treatment as described herein infra. It is significant to note that once the membranes are altered to the fusogenic state in accordance with the present invention, all that is then required to induce fusion per se is simply to bring the fusogenic membranes in contact with each other by any suitable means e.g., centrifugation, packing by increasing density, exposure to pulses of alternating current and the like, whereby fusion takes place spontaneously without any further treatment. Furthermore, the fusogenic membranes could be held in this altered state for substantial periods of time up to several minutes prior to fusion, which may allow various manipulations of these fusogenic membranes, e.g. by exposure to chemicals, pharmaceuticals, microorganisms, gene-altering substances and the like. Other advantages of the invention would be apparent to those skilled in the art.
2. Prior Art
Fusion has been induced in many membrane systems using procedures involving electric fields. The state of the art has been reviewed by Zimmermann et al. 1983, Biotechniques 1, 118-122; Crane 1983, American Biotechnology Laboratory 1, 74-79; Zimmermann et al. 1982, J. Biol. Phys. 10, 43-50; and Zimmermann 1982, Biochim. Biophys. Acta. 694, 227-227, which are incorporated herein by reference. In general, membranes in suspension are fused by first exposing the suspension to a lower strength alternating current (AC) which causes the membranes to come in close proximity with each other as they line up in the so-called "pearl-chain" formation. This phenomenon has been named dielectrophoresis. (Zimmermann et al. 1982, J. Memb. Biol. 67, 165-182). Fusion is then induced by the application of a higher strength direct current (DC) pulse.
The present invention is quite different from the prior art techniques. In accordance with the present invention, a fusogenic state can be created in separate and individual biological membranes by a treatment with exponentially decaying electric field pulses. The membrane fusion properties of this state are fundamentally different from currently understood membrane fusion phenomenology using electric fields.
Although in a certain aspect of the process, the prior art and the present invention both use pulses which have similar electric field strengths in the membrane suspension medium (500-1000 V/mm), the quality and characteristics of the pulse waveform used is quite different. Whereas the prior art uses a square waveform, i.e., a very rapid risetime and a very rapid fall time, the present invention uses an exponentially-decaying pulse, i.e., a very rapid risetime and an exponentially-decaying fall time. Hence, even in this respect the timescale or "length" of the two different kinds of pulses are not comparable.
Membrane fusion and fusion-associated phenomena are of interest because of their involvement in biological processes. However, the mechanism of fusion is not fully understood. Therefore, creating specific experimental conditions so as to reproducibly manipulate or control the membrane fusion process is not currently possible. For example, fusing membranes to mix membrane components having different origins or for delivery of certain substances to a recipient cytoplasm or internal aqueous compartment cannot be always accomplished by the present state of the art for the simple reason that a delayed interval of time for holding the altered membranes in a fusogenic state was not heretofore possible.
The present invention offers an improved device and a process for inducing fusogenic state which allows fusion and its manipulation in a manner not heretofore possible.